Coulometric Karl Fischer (KF) titration is a highly accurate technique employed for moisture determination in non-aqueous solvents. Methodology for coulometric titration in a KF apparatus coupled to an autosampler oven was optimized in order to improve the accuracy of moisture determination in biodiesel derived from soybean. Several parameters were investigated, and three of them were optimized according to a central composite 23 star experimental design. The highest accuracy for moisture determinations corresponded to a relative standard deviation of 1.48 %. It was reached when the KF titrator was operated with electrode without diaphragm, the sample mass was 4 g, and the oven temperature was set to 170°C. Other parameters, like carrier gas flux and extraction time, presented minimal or no effects for improving the accuracy of moisture determination.